Yozo Mitsui, Department of Urology, Shimane University School of Medicine, Izumo 693-8501, Japan. e-mail: email@example.com
What's known on the subject? and What does the study add?
The process of bladder regeneration with a bladder acellular matrix graft (BAMG) is thought to be accelerated by administration of vascular endothelial growth factor into the host bladder.
In the present study, we showed that simultaneous implantation of bilateral ureters into a BAMG after a partial cystectomy is reasonable and provides an increased opportunity to the bio-scaffold for communication with host tissues from which a blood supply and stem cells will be generated.
• To evaluate if the implantation of bilateral ureters into a bladder acellular matrix graft (BAMG) at the time of its implantation would enhance bladder regeneration in a partial substitution BAMG.
MATERIALS AND METHODS
• Partial cystectomies were performed under general anaesthesia in 12 pigs, followed by augmentation with a BAMG.
• Six (ureteric implantation group) also received simultaneous implantation of bilateral ureters into the BAMG, while the remaining six (control group) did not have ureteric implantation.
• In both groups, bladder regeneration was evaluated using endoscopic and histopathological methods at 1, 2, 4, and 8 weeks after implantation.
• At 1 week after BAMG implantation, there were significant inflammatory changes on the host bladder in both groups, while no significant endoscopic changes were seen on the BAMG luminal surfaces.
• At 2 weeks, inflammatory changes were diminished and epithelialisation on the BMAG was identified, especially near the host bladder in both groups.
• Similarly, epithelialisation on the BAMG near the implanted ureters was seen in the ureteric implantation group.
• At 4 and 8 weeks, epithelialisation remained in progress in both groups, although it was more active and expansive in the ureteric implantation group.
• In our porcine model, endoscopic and histopathological examinations showed that simultaneous implantation of bilateral ureters into a BAMG enhanced epithelialisation of the AMG.
• This new approach using host ureters and bladder as a potential source of bladder regeneration may provide for rapid and complete regeneration of a bladder substitute.
In cases with severe bladder dysfunction caused by urological disorders, both congenital and acquired, intestinal substitution or augmentation is a frequent approach to treat the condition. In such cases, issues of concern included those related to complications brought about by intestinal replacement, e.g. occurrence of metabolic disorder, acceleration of intestinal mucus product, calculi formation, infection, and malignant transformation of intestinal ‘bladder’ mucosa [1–3]. Furthermore, patients who undergo an abdominal operation are probably limited in regard to its use for application of a replacement. In 1990, Langer and Vacanti  advocated the basic concept of ‘tissue engineering’, in which a scaffold implanted with cells applied to an organ is absorbed and replaced with an extracellular matrix induced by the host cells, indicating the necessity of absorbable scaffolds of high quality as an important prerequisite in tissue engineering strategies.
We have shown the feasibility and reproducibility of acellular matrix grafts (AMGs) as acceptable bio-scaffolds in small animals . Even in hosts with a diseased bladder, we have conducted bladder regeneration in a way that could be completed by a combination of urothelium, muscle fibres, and nerve regeneration in a significant and proper direction [5–7]. However, those results were obtained in small animal studies. To validate the feasibility and clinical application of AMGs, studies with larger animals are required. Likewise, a more complete and prompt method of bladder regeneration would be desirable for clinical applications.
We have also reported that the process of bladder regeneration with a bladder AMG (BAMG) was accelerated by administration of vascular endothelial growth factor into the host bladder . If an increased blood supply and source of regeneration can be secured around an implanted BAMG, the chance of rapid and complete process of bladder regeneration will be enhanced. To pursue bladder regeneration in a complete way, blood supply and stem cells for regeneration should both come from host tissues. Thus, a greater abundance of host tissues or cells may help to regenerate the bladder in a prompt and complete manner.
To investigate the possibility of easy and prompt bladder regeneration with an increased opportunity of stem cell migration into the AMG, we established a porcine model that used simultaneous implantation of bilateral ureters into a BAMG after a partial cystectomy.
MATERIALS AND METHODS
PREPARATION OF HOMOLOGOUS BAMG
Fresh bladders with the bilateral ureters were harvested from pigs, and homologous bladder materials were used for preparation of BAMGs. Each obtained bladder was incubated with DNase type I at 37 °C for 7–10 days. After completing the process of digestion with DNase type I, the bladder materials were incubated with sodium deoxycholate at 37 °C and observed for 2–3 days. When the bladder material had changed in colour to white, the process was considered to be close to completion, and completely processed BAMGs were preserved at 4 °C in 1% neomycin solution containing 1% amphotericin B. To confirm the complete process of BAMG formation, in which no cellular competent should be present except for collagen fibres, haematoxylin and eosin (H&E) staining was performed.
HOMOLOGOUS BAMG IMPLANTATION AFTER PARTIAL CYSTECTOMY
We performed two experiments based on (A) the notion that application of BAMG in a large animal should be performed to confirm its feasibility as a scaffold for bladder regeneration and (B) our hypothesis that implantation of bilateral ureters into a BAMG during its implantation would accelerate the process of bladder regeneration.
The first experiment (A) was performed as a preliminary study and two female pigs with a body weight of ≈20 kg were used. Before intubation, ketamine at 20 mg/kg was administered. For anaesthesia, induction and maintenance was performed using an i.v. injection of pentobarbital at 5 mg/kg. When anaesthesia was sufficient, a lower abdominal skin incision was made, the prevesical space was identified and further developed, and a 50% partial cystectomy was performed. A prepared BAMG sized 7 × 7 cm was implanted into the bladder, which had undergone a partial cystectomy, with four marker sutures of 5-0 nylon placed at four points, with in-between running sutures of 5-0 monofilament absorbable polyglyconate (Maxon®) in two-layers to make an anastomosis between the BAMG and host bladder (Fig. 1).
Experiment B was performed to test our hypothesis and 12 female pigs with a body weight of ≈20 kg were used. Six of the pigs were used as a control group, in which a BAMG was implanted into the host bladder after a 50% partial cystectomy. In the remaining six pigs, implantation of the bilateral ureters into the BAMG was performed at the same time as its implantation. After the partial cystectomy, the distal ends of the bilateral ureters were implanted into the BAMG using a running suture composed of 5-0 polyglactin 910 (Vicryl®). Next, an anastomosis was made between the host bladder and BAMG using 4-0 absorbable suture in a two-layer fashion (Fig. 1). All experiments were approved by the Animal Care Committee, Shimane University School of Medicine (Protocol #H17-117).
VALIDATION OF BLADDER REGENERATION
In experiment A, the bladders were harvested 2 months after BAMG implantation for histopathological evaluation. In experiment B, detailed endoscopic examinations were performed at 1, 2, 4, and 8 weeks after BAMG implantation to validate bladder regeneration on the scaffold. Findings from the endoscopic examinations were used to evaluate the differences in the process of inner epithelialisation between the BAMG and host bladder. In addition, a cold cup biopsy was taken at 4 weeks after BAMG implantation to avoid fear and tenderness in the pigs. At 8 weeks after BAMG implantation, the bladders were harvested for immunohistochemical analysis to determine muscle fibre development and neo-vascularity. In both experiments, sufficient anaesthesia comprised of ketamine at 20 mg/kg and i.v. pentobarbital at 5 mg/kg were provided during the procedures.
Obtained whole bladder tissues were fixed on a board with buffer formalin. Formalin-fixed paraffin-embedded materials were used for histopathological evaluation, with sections sliced at 5 µm for H&E staining and immunostaining. To determine muscle fibre development and neo-vascularity, an anti-human α-actin (Dako, Japan) monoclonal antibody was used. A conventional approach to detect immunoreactivity was applied using a commercially available Envison System kit (Dako, Japan). To determine the presence of nerve fibres, an anti-human protein gene product 9.5 (PGP9.5) monoclonal antibody was used with an antigen retrieval technique. Briefly, paraffin embedded 5-µm thick sections were subjected to deparaffinization and subsequent hydration. After blocking endogenous peroxidase activity with 3% H2O2 solution, the processed sections were stained with anti-human PGP9.5 antibody after applying an antigen retrieval technique with 10 mm citrate buffer (pH 6.0). The following step was performed using the commercially available EnVision system (Dako, Japan) with 3,3′-diaminobenzidine (DAB) as the chromogen (Dako, Japan). Meyer's haematoxylin solution was used for counterstaining.
BLADDER REGENERATION WITH HOMOLOGOUS BAMG
In experiment A, we investigated whether a homologous BAMG can be used to regenerate bladder tissue in vivo. At 8 weeks after BAMG implantation there was no difference in histological structure between the host bladder wall and the BAMG near the host bladder. In the central region of the implanted BAMG, muscle fibre organisation was on-going (Fig. 2).
BAMG IMPLANTATION-RELATED COMPLICATIONS
In experiment B, four of the pigs in the control group, with BAMG implantation alone, survived for 8 weeks, while one died on postoperative day (POD) 2 (respiratory arrest) and one on POD 19 (graft rejection). In the experimental group, with simultaneous implantation of bilateral ureters, four pigs survived for 8 weeks, while one died on POD 2 from a UTI and one on POD 6 of renal failure. Cystoscopic examinations were performed in both groups under sufficient general anaesthesia at 1, 2, 4, and 8 weeks after the operation. Thus, at 8 weeks after implantation four pigs from each group were analysed for histological differences in bladder regeneration.
ENDOSCOPIC AND HISTOPATHOLOGICAL EVALUATIONS OF BLADDER REGENERATION
Endoscopic examination findings in experiment B at each stage after BAMG implantation are shown in Fig. 3. At 1 week after implantation, there were significant inflammatory changes in the host bladder in both groups, while no significant endoscopic changes were found on the luminal surface. At 2 weeks after implantation, the inflammatory changes had diminished and epithelialisation on the BMAGs was identified, especially near the host bladder, in both groups. Likewise, epithelialisation on the BAMGs near the implanted ureters was found in the ureteric implantation group. At 4 weeks after BAMG implantation, epithelialisation on the BAMGs continued in both groups, although more active and expansive epithelisation was found in the ureteric implantation group. At 8 weeks after implantation, the endoscopic findings noted at 4 weeks were more evident in the ureteric implantation group as compared with the control group. Histological findings related to bladder regeneration after 4 weeks are shown in Fig. 4. In the area of the BAMG near the host bladder, neo-vascularity was more abundant in the ureteric implantation group, while several small vessels were found on the portion of the BAMG near the ureter.
IMMUNOHISTOCHEMICAL EVALUATION OF BLADDER REGENERATION
Results of immunostaining of α-actin and PGP9.5 at 8 weeks after BAMG implantation in experiment B are shown in Fig. 5. In the control group, epithelialisation completely covered the entire surface of the BAMG. Even in the central portion of the implanted BAMG, neo-vascularity was found, although α-actin positive muscle fibres were scarce, while PGP9.5-positive nerve fibres were found even in the central portion of the BAMG. In the ureteric implantation group, the same trend seen in the control group was noted, while in the central portion of the BAMG, there was greater vascularity in the ureteric implantation group than in the control group. The major difference in immunostaining findings between the two groups was in the distribution of α-actin-positive muscle fibres. Both muscle fibres and neo-vascularity were more abundant in the ureteric implantation group, and those were more evident in BAMG portions close to the host bladder as well as adjacent to the implanted ureters. PGP9.5-positive nerve fibres were also found in the central portion of the BAMG in that group.
The major drawback of bladder substitution using the small intestine is essentially associated with functional impairment of the neobladder, as storage-related function is excellent, while void function is far from complete. A scaffold made from bio-materials is considered to be an excellent candidate for regenerating a functional bladder in a complete and organised fashion . Previous studies using small animals, e.g. rodents, clearly demonstrated complete bladder regeneration using homologous BAMG implantation, even in hosts with diseased bladders [5–7]. Considering the clinical feasibility of BAMG-based bladder regeneration, experiments with larger animals, e.g. pigs, are important.
In a dog model, implantation of a BAMG after a partial cystectomy with 20–50% removal of the host bladder showed that epithelialisation of the inner surface of the BAMG was completely processed within 1 month after implantation and the process of muscle fibre regeneration was probably delayed, especially around the central portion of the implanted BAMG, which took at least 7 months to be completely processed . In addition, the implanted BAMG probably underwent shrinkage and was decreased down to 70% of the host bladder. Therefore, emphasis should be placed on graft shrinkage from an incomplete and/or delayed process of muscle regeneration in the BAMG, which must be avoided for clinical feasibility.
In another experiment with dogs using a small intestinal submucosa (SIS) graft, Badylak et al.  reported that vascular regeneration and epithelial covering were probably finished by the end of 4 weeks after implantation, Thereafter, muscle development with its accompanying organisation was found at 8 weeks after the operation and the implanted SIS graft was completely substituted with regenerated bladder tissue at the end of 12 weeks. These alterations were inversely correlated with reduced amounts of collagen fibres and infiltration of inflammatory cells within the scaffold. In another dog model, SIS implantation into hosts with bladders reduced by 45% resulted in no scar formation related to graft shrinkage up to 15 months after graft implantation . By contrast, it should be noted that a partial cystectomy of 90% of the host bladder coupled with subsequent SIS grafting 1 month later did not result in bladder regeneration .
We consider that larger tissues subjected to regeneration will show greater delays in the processes of neo-vascularity and nerve regeneration. In addition, delayed tissue regeneration will surely result in early tissue degeneration before regeneration is completed, possibility resulting in active inhibition of tissue regeneration, such as persistent inflammation-related scar formation within the BAMG.
Based on these observations, we consider that an abundant blood supply coupled with stem cells produced by the host tissue will activate the process of tissue regeneration. We hypothesised that implantation of bilateral ureters into a BAMG at the time of its implantation after a partial cystectomy would accelerate bladder regeneration. As shown in Fig. 3, at both 4 and 8 weeks after BAMG implantation, a more active and expansive pattern of epithelialisation was endoscopically evident on the inner surface of the implanted BAMGs in the ureteric implantation group as compared with those in the control group. Likewise, at 8 weeks after implantation, muscle fibres and neo-vascularity were more abundant in the ureteric implantation group (Fig. 5). Importantly, increased muscle fibres and neo-vascularity were significant in the region near the host bladder and also that adjacent to the implanted ureters. In addition, PGP9.5-positive nerve fibres were detected in the central portion of the BAMG. Together, these findings suggest that simultaneous implantation of bilateral ureters into the BAMGs contributed to beneficial effects, including prompting and expanding bladder regeneration.
Major concerns about application of a scaffold made from bio-materials include whether the scaffold will become an active inducer of significant inflammation, which would avert the normal process of regeneration and finally result in severe shrinkage of the regenerated BAMG as the bio-materials become absorbed by the host cells. Thus, it is important to note that these negative effects on tissue regeneration can be prevented and resolved if bladder regeneration can be properly processed and finished by the time the host cells completely absorb the bio-scaffold. Previous studies have reported prevention of early and persistent activation of the degenerative pathway associated with tissue re-modelling in BAMG experiments, including extrinsic cytokine injection into the host bladder , as well as implantation of cytokines in a depot preparation into the BAMG or usage of the BAMG after immersion in a cytokine solution before the operation .
In a future study, identification of cytokine families actively involved in the process of bladder regeneration may overcome the potential drawbacks of bio-scaffolds such as BAMG, so that bladder regeneration can be promptly and precisely processed and completed. In the present study, we applied bilateral ureteric implantation simultaneously with implantation of the BAMG for the purpose of making a wide contact with host tissue, from which blood supply and stem cells could be supplied, subsequently accelerating and expanding bladder regeneration. This may be why in previous investigations that used a BAMG, emphasis was placed on covering the scaffold quickly, as it has been reported that cells cannot survive within tissue that is >0.8 mm thick without an appropriate blood supply . At the very early stage of tissue regeneration, neo-vascularity is essential for producing an appropriate environment to promote and activate ingrowth of migrated cells within the scaffold .
The concept of implantation of bilateral ureters into a BAMG at the time of its implantation after a partial cystectomy seems reasonable for providing an increased opportunity to the bio-scaffold for communication with host tissues, from which a blood supply and stem cells will be generated. The present findings showed the feasibility of using a BAMG as a bio-scaffold even in large animals to regenerate bladder tissue. The present operative procedures are thought to be improved in such a way that the site of ureteric implantation is the best candidate for maximal connection between host tissue and the implanted BAMG.